Committee Member

Committee Member

Committee Member

Keywords

Abstract

Stroke is the fifth leading cause of death in the United States and a major cause of long-term disability in industrialized countries. The core region of an ischemic stroke dies within minutes due to activation of necrotic pathways. Outside of this core region is the penumbral zone, where some perfusion is maintained via collateral arteries. Delayed cell death occurs in this area due to the triggering of apoptotic mechanisms, which expands the ischemic injury over time. The cellular and molecular events that produce the expansion of the ischemic core continue to be poorly understood. The increases in the amyloid precursor protein and pathogenic secretases lead to the increase in amyloid-β (Aβ) production. The relatively small amount of research in this area has hampered development of stroke therapy designed to prevent neuronal and glial cell degeneration in the penumbra. Currently, there is a significant lack of therapeutic options for acute ischemic stroke, and no drug has been approved for treating patients at delayed time points (≥ 4.5 hr post-stroke).

Afobazole, an anxiolytic currently used clinically in Russia, has been shown to reduce neuronal and glial cell injury in vitro following ischemia, both of which have been shown to play important roles following an ischemic stroke. Treatment with afobazole decreased microglial activation in response to ATP and Aβ, as indicated by reduced membrane ruffling and cell migration. Prolonged exposure of microglia to ischemia or Aβ conditions resulted in glial cell death that was associated with increased expression of the pro-apoptotic protein, Bax, the death protease, caspase-3 and a reduced expression in Bcl-2. Co-application of afobazole decreased the number of cells expressing both Bax and caspase-3, while increasing the cells expressing Bcl-2 resulting in a concomitant enhancement in cell survival. While afobazole inhibited activation of microglia cells by Aβ25-35, it preserved normal functional responses in these cells following exposure to the amyloid peptide. Intracellular calcium increases induced by ATP were depressed in microglia after 24 hr exposure to Aβ25-35. However, co-incubation with afobazole returned these responses to near control levels. Therefore, stimulation of sigma-1 and sigma-2 receptors by afobazole prevents Aβ25-35 activation of microglia and inhibits Aβ25-35-associated cytotoxicity.

Examining the molecular mechanisms involved in the increased neuronal survival demonstrates that ischemia or Aβ results in an increased expression of the pro-apoptotic protein Bax and the death protease caspase-3, while at the same time decreasing expression of the anti-apoptotic protein, Bcl-2. However, unlike observations made with microglia, afobazole was unable to modulate this ischemia-induced expression, but was able to modulate Aβ-induced expression of apoptotic proteins while still rescuing neurons from death. Additional experiments were carried out to understand this disparity between the failures of afobazole to prevent the up-regulation of pro-apoptotic genes while retaining the ability to mitigate neuronal death. Although the neurons were still alive they were in a senescent state and were unresponsive to depolarization by high K+. However, these findings are still positive due to the ability of afobazole to delay neuron death, thus minimalizing the toxic environment of the penumbra.

These comorbidities of ischemia and Aβ toxicity may lead to potentiated responses and increase the risk for various vascular dementias. It was of particular interest to study how the convergence of ischemia, acidosis and Aβ influence cellular activity and survival within core and penumbral regions. Application of Aβ increased the [Ca2+]i overload produced by concurrent ischemia + acidosis application in isolated cortical neurons. We found that the acid-sensing ion channels 1a (ASIC1a) are involved in the potentiation of [Ca2+]i overload induced by Aβ. Furthermore, afobazole (100 uM) abolished Aβ potentiation of ischemia + acidosis evoked [Ca2+]i overload, which may represent a therapeutic strategy for mitigating injury produced by Aβ and stroke.